Enzymatic resolution of aryl and thio-substituted acids

ABSTRACT

Provided is a method of resolving a racemic mixture of a compound of formula I to obtain a desired enantiomer:  
                 
 
     wherein Ar is C 6  or C 10  aromatic group that can be substituted with H, C 1  to C 6  alkyl, trifluoromethyl or halo, R 5  is halo or —S—R 1 , wherein R 1  is H or acetyl, and R 2  is H or C 1  to C 6  alkyl, the method comprising: reacting a compound of formula I wherein the compound is an ester whereby R 2  is C 1  to C 6  alkyl with a lipase derived from Mucor meihei to stereoselectively hydrolyze the ester bond to produce an acid; and isolating the acid, wherein the reaction is conducted in a solvent comprising 80% to 98% v/v % organic phase and a residue of water phase (which can be buffered).

[0001] This application claims priority from U.S. Application No.60/233,193 filed Sep. 15, 2000.

BACKGROUND OF THE INVENTION

[0002] Over the last several years compounds have been reported in thepatent and technical literature as possessing angiotensin convertingenzyme (ACE) inhibitory activity or neutral endopeptidase (EC 3.4.24.11;NEP) inhibitory activity. Additional compounds have been identified thatpossess both inhibitory activities. These dual inhibitor compounds areof interest as cardiovascular agents particularly in the treatment ofhypertension, congestive heart failure, and renal disease. Thesecompounds are also referred to as vasopeptidase, dual metalloprotease,NEP/ACE, or ACE/NEP inhibitors.

[0003] Omapatrilat is such a vasopeptidase inhibitor which is currentlyundergoing clinical evaluation. Omapatrilat has the chemical name[4S-[4α(R*), 7α,10aβ]]-octahydro-4-[(2-mercapto-1-oxo-3-phenylpropyl)amino]-5-oxo-7H-pyrido[2,1-b][1,3]thiazepine-7-carboxylic acid and the structural formula:

[0004] Omapatrilat, its preparation, and its use in treatingcardiovascular disease are disclosed by Robl in U.S. Pat. No. 5,508,272.

[0005] Gemopatrilat having the chemical name [S—(R*,R*)]-hexahydro-6-[(2-mercapto-1-oxo-3-phenylpropyl)amino]-2,2-dimethyl-7-oxo-1H-azepine-1-aceticacid is another vasopeptidase inhibitor which is currently undergoingclinical evaluation. This compound has the structural formula:

[0006] This compound, its preparation, and its use in treatingcardiovascular diseases are disclosed by Karanewsky et al. in U.S. Pat.No. 5,552,397. Processes for preparing vasopeptidase inhibitorsincluding omapatrilat and gemopatrilat are disclosed by Kronenthal et alin WO 99/35145 and processes for preparing omapatrilat and omapatrilatintermediates are disclosed by Godfrey et al in U.S. Pat. Nos. 6,166,227and 6,248,882, by Moniot et al. in U.S. Pat. No. 6,162,913, by Hanson etal. in U.S. Pat. No. 6,140,088, and by Patel et al. in WO 00/14265.Other processes for preparing omapatrilat intermediates are disclosed byTaoka et al. in U.S. Pat. No. 6,174,707, by Tanaka et al. in U.S. Pat.No. 6,174,711, and by Boesten et al. in U.S. Pat. Nos. 6,133,002 and6,222,052.

[0007] Methods for producing thio aryl and thio-substituted acidmoieties such as those incorporated into such vasopeptidase inhibitorsare costly. For example, unnatural amino acids such as D-phenylalaninecan be converted to α-halides with retention of configuration (usingdiazotization conditions) and stereospecifically inverted to anappropriate thio-containing intermediate. However, the cost ofenantiomerically pure D-phenylalanine is high. Methods are needed thatproduce the desired enantiomer from inexpensive starting materials.

[0008] Methods of using lipases in aqueous buffers to stereoselectivelyproduce a desired acid from 2-acetylthio-3-phenylpropionic acid esterhave been described in JP2000-23693A. However, usable e.e. values arenot obtained with a particularly desirable enzyme, the lipase obtainedfrom Mucor Meihei and marketed by Novo Nordisk Biotech, Inc. (soon to beNovozymes, Inc.) as Lipozyme IM. An improved method is needed to obtainhigher e.e. values and to allow the reuse of the enzyme.

[0009] Furthermore, the art contains no detail on how to purify thecrude product from stereoselection using the lipozyme IM enzyme to aproduct that would be suitable for pharmaceutical use (>98.5% ee). Amethod is needed for this.

[0010] Also, there is no demonstration of how to reuse the recoveredunreacted ester. If the recovered ester is not reused, then the costwill be prohibitive. Methods for the reuse of the unreacted ester arealso desired.

SUMMARY OF THE INVENTION

[0011] The invention provides a method of resolving a racemic mixture ofa compound of formula I to obtain a desired enantiomer:

[0012] wherein Ar is C₆ or C₁₀ aromatic group that can be substitutedwith H, C₁ to C₆ alkyl, trifluoromethyl or halo, R₅ is halo or —S—R₁,wherein R₁ is H or acetyl, and R₂ is H or C₁ to C₆ alkyl, the methodcomprising: reacting a compound of formula I wherein the compound is anester whereby R₂ is C₁ to C₆ alkyl with a lipase derived from Mucormeihei to stereoselectively hydrolyze the ester bond to produce an acid;and isolating the acid, wherein the reaction is conducted in a solventcomprising 80% to 98% v/v % organic phase and a residue of water phase(which can be buffered).

[0013] Another aspect of the invention provides a method ofstereoselectively producing a desired enantiomer of a compound offormula I:

[0014] wherein Ar is C₆ or C₁₀ aromatic group that can be substitutedwith H, C₁ to C₆ alkyl, trifluoromethyl or halo, R₁ is H or acetyl, andR₂ is H or C₁ to C₆ alkyl, the method comprising: reacting Ar—CH₂—X,where X is a leaving group, with R₄—C(O)—CH₂—C(O)O—R_(2*), whereinR_(2*) and R₄ are independently C₁ to C₆ alkyl; reacting a resultingcompound of formula II:

[0015] with a halogenating agent which comprises an N-halo substitutedamide, N-halosubstituted imide, N-halosubstituted thioamide, orN-halosubstituted thioimide as the halogenating moiety to produce, withor without an additional hydrolysis of the ester, a compound of formulaIII:

[0016] wherein Y is the leaving group; reacting the compound of formulaIII with Z—S—R₁*, wherein R₁* is acetyl, and Z is K, Na, or other cationto produce a compound of formula I*: and

[0017] conducting one of the following stereoselective reactions: (a)(1) reacting the compound of formula III with a hydrolase that isstereoselective for the ester; (2) isolating the desired resulting acid;(3) racemizing residual compound of formula III; and (4) conducting atleast one additional iteration of steps (a)(1) and (a)(2) with theracemized residual compound of formula III, wherein the reacting withZ—S—R₁* is conducted with stereoselective inversion of the chiralcarbon; or (b) (1) reacting the compound of formula I* with a hydrolasethat is stereoselective for the ester; (2) isolating the desiredresulting acid; (3) racemizing residual compound of formula I*; and (4)conducting at least one additional iteration of steps b(1) and b(2) withthe residual racemized compound of formula I*.

[0018] The method may further comprise: crystallizing the compound offormula I* to obtain the compound of formula I* in increasedenantiomeric purity. In a preferred embodiment the isomeric purity ofthe compound of formula I* is at least 98% ee.

[0019] The method may further include reacting with a catalytic amountof tetraalkylammonium halide in the racemization steps of a(3) and b(3).

[0020] In one preferred embodiment of the method, the halogenating agentis N,N-dibromo-5,5-dimethylhydantoin. In another preferred embodimentthe halogenating agent is N,N-dichloro-5,5-dimethylhydantoin.

[0021] In a further aspect, the invention provides a method of preparinga compound of formula II:

[0022] wherein R₂ and R₄ are independently C₁ to C₆ alkyl, the methodcomprising: reacting at least five equivalents of R₄—C(O)—CH₂—C(O)O—R₂with ArCH₂Cl wherein Ar is C₆ or C₁₀ aromatic group that can besubstituted with C₁ to C₆ alkyl or halo, wherein the reaction isconducted in a solution consisting essentially of the reactants and nomore than 1.2 molar equivalents of a base source of sodium or potassiumC₂ to C₆ alkoxide, which can be provided in the corresponding alcohol.In a preferred embodiment the alkoxide concentration in the base sourceis at least 3 M.

DETAILED DESCRIPTION OF THE INVENTION

[0023] This invention relates to the methods of stereoselectivelyproducing desired enantiomers of important intermediates for producingpharmaceuticals.

[0024] The invention relates to a method for the enzymatic resolution ofaryl and thiosubstituted acids. Additionally, a process for thepreparation of the enzyme substrate aryl and thiosubstituted acidderivatives are disclosed.

[0025] The methods of the invention are described with reference toformulas I, I*, II and III, as outlined above in the Summary. Ar is C₆or C₁₀ aromatic group that can be substituted with H, C₁ to C₆ alkyl(preferably C₁ to C₃), trifluoromethyl or halo, R₅ is halo or —S—R₁,wherein R₁ is H or acetyl, and R₂ is H or C₁ to C₆ alkyl (preferably C₁to C₃). R₄ is C₁ to C₆ alkyl (preferably C₁ to C₃). R_(2*) is C₁ to C₆alkyl (preferably C₁ to C₃).

[0026] The invention can be described with reference to Scheme 1.

[0027] An overall process for preparing, for example,3-aryl-2-acetylthio-substituted propanoic acids (1) is illustrated inScheme 1. Initially, alkyl acetoacetates (or any other β-keto esters)are alkylated with benzyl chloride (2) (or substituted analogs thereof)to provide keto esters (3). A halogenation-deacetylation reactioneffects the conversion of 3 to the 2-halo-3-arylpropanoic esters (4).Direct treatment of 4 with thioacetate salts furnishes racemic alkyl2-acetylthio-3-aryl propanoates (5). At this stage, an enzymaticresolution of 5 can be effected. Enantioselective hydrolysis of theester of 5, for example, provides the S-isomer of acid 1 with excellentenantioselectivity. 1 can be conveniently separated from the unreactedR-ester 5R. The invention also provides for methods for recycling of theunreacted R ester (5R) to provide additional supplies of the racemicsubstrate for the enzymatic resolution step. In an alternativeembodiment of the invention, the enzymatic resolution step is carriedout at the stage of the α-halo ester 4. In this case enzymes andreaction conditions are chosen to optimize the enantioselectivehydrolysis of the R-halo ester and provide R-halo acid (6). 6 can bedisplaced with inversion of configuration with a thioacetate salt toprovide 1. Again a method is provided for recycling unreacted ester (4S)from the enzymatic resolution step to provide additional racemicsubstrate for the hydrolysis reaction.

[0028] Alkyl 2-aryl methylacetoacetates (3) can be prepared convenientlyand in high yield by alkylation of alkyl acetoacetates using optimizedconditions that Applicants have developed. Using ethyl acetoacetate andbenzyl chloride, Applicants have found that the presence of excess ethylacetoacetate, without any added solvent (other than the ethanol that isin the concentrated commercially available reagent sodium ethoxidesolution) afforded high yields and exclusive mono C-alkylationselectivity. Other methods for conducting this reaction generallyrequire the use of a catalyst (Brandstrom, A. and Junggren, U. ActaChem. Scand. 1969, 23, 2204; Durst, H. D. and Liebeskind, L. J. Org.Chem., 1974, 39, 3271) or use of an inorganic support (Ranu, B. C. andBhar, S. J. J. Chem. Soc. Perkin Trans. 1992, 1, 365). In addition, thepreferred methods used in the invention avoids one or more limitationssuch as the use of toxic materials, laborious and time-consumingprocedures, and/or relatively low yields of products. For example, in anon-preferred reaction of ethyl acetoacetate and benzyl chloride,without a phase transfer catalyst, the alkylation reaction provided amodest 25% yield after refluxing in benzene for 8 hours (Durst, H. D.and Liebeskind, L. J. Org. Chem., 1974, 39, 3271). The preferredreaction conditions of the invention allow the utilization of lessreactive benzyl chlorides to provide high yields. It is advantageous touse less expensive benzyl chlorides versus more expensive benzylatingagents, such as benzyl bromides (and, in general aryl methyl chloridesversus aryl methyl bromides), as starting materials in large scalemanufacturing.

[0029] Suitable bases for the alkylation reaction include sodium,potassium, or lithium alkoxides, such as sodium ethoxide, K₂CO₃ or NaH.The corresponding sodium, potassium, or lithium alkoxides of the alkylester are preferred bases for the reaction to avoid thetransesterification reactions that can plague reactions of esters. Oftena convenient source of alkali metal alkoxide bases are in solutions ofthe corresponding alcohol that are either commercially available orreadily prepared. These alkoxides include sodium, potassium, or lithiumsalts of C₁ to C₆ alkoxides. Preferably the concentration of this basesource solution is at least 3 M. A slight molar excess of base, such as1.2 molar equivalents relative to the aryl methyl chloride is used.

[0030] It can be recognized by those in the art that these reactionconditions are useful for other alkyl β-ketoesters of the formulaR₄—C(O)—CH₂—C(O)O—R₂, besides ethyl acetoacetate. These β-ketoestersinclude compounds where R₄ and R₂ are independently C₁-C₆ alkyl, orpreferably C₁ to C₃ alkyl. Moreover, in addition to benzyl chlorideother arylmethyl chlorides can be used including C₆ or C ₀ alkyl or halosubstituted arylmethyl chloride groups. Moreover the aryl moiety can besubstituted by H, C₁ to C₆ alkyl or halo groups. These aryl methylchloride groups include, for example, benzyl and napthylmethyl chlorideanalogs. A large molar excess of the alkyl acetoacetate relative to thearylmethyl chloride is preferably used, preferably at least a 5 foldexcess. The excess of starting acetoacetate can, for example, beconveniently recovered by fractional distillation under reducedpressure, and reused in the process. The yield of the alkylationreaction is at least 95%, more preferably at least 99%.

[0031] Keto ester 3 is converted to α-halo ester 4 using ahalogenation/deacetylation sequence. Bromination-deacetylation reactionof ethyl 2-benzylacetoacetate to give 2-bromo-3-benzenepropanoate hasbeen reported using N-halo succinimides. Applicants have discovered thatthe expensive brominating agent, N-bromosuccinimide (NBS), can beeffectively replaced by a cheaper reagentN,N-dibromo-5,5-dimethylhydantoin (DBDMH) in this reaction. In additionto its low price, both bromine atoms are used in thebromination-deacetylation transformation to further contribute to itscost-effectiveness. A comparable yield can be achieved using DBDMH inthe reaction as when NBS is used. The product 4 can be convenientlyisolated from the reaction mixture by, for example, vacuum distillation.The yield of the reaction is preferably at least 80%.

[0032] Similarly, it can be recognized by those in the art thatN,N,-dichloro-5,5-dimethylhydantoin (DCDMH) can replaceN-chlorosuccimide in reactions generating the corresponding α-chloroproducts. The use of chlorinating agent, DCDMH, achieves the sameeconomic advantages of the corresponding brominating agent, DBDMH. OtherN-halo-amides, -imides, -thioamides, or -thioimides (including cyclichalo-amides, -imides, thioamides, or thioimides) effective to donate thehalo moiety can also be used. Preferred N-halo-amides, -imides,-thioamides, or thioimides (including cyclic halo-amides, -imides,thioamides, or thioimides) are those including the moiety—C(O)—N(X)—C(O)—N(X)—, where the halo moiety, X, is chloro or bromo.

[0033] α-Halo esters 4 can be converted to the (x-acetylthio esterintermediate 5 using thioacetate salts. Thioalkanoyl salts containingany cation can be used to effect the transformation including ammonium(including alkylammonium) salts or metal salts. Metal alkali salts suchas sodium and potassium salts are preferred salts.

[0034] The intermediate ester 5 can be hydrolyzed enantioselectively tofurnish the S-acid 1 along with unreacted R-ester 5R using an enzymatichydrolysis procedure. In one embodiment, the lipase is immobilized onparticles of a solid support. Mucor genus derived lipases (such asLipozyme IM from Novo Nordisk Ltd.) have been found to be particularlywell-suited for the hydrolysis reaction with this substrate. Using thisenzyme and the optimized reaction conditions found by Applicants, it ispossible to obtain enantiomeric excess's (e.e.'s) of, for example, 96%or better with high product yields of, for example, 75% or better (basedon the consumed ester).

[0035] Besides enzyme selection, other reaction conditions are alsoimportant for high enantioselectivity in the enzymatic resolution. Thereaction conditions can be altered by several variables includingsubstrate concentration, solvent, pH, and incubation time. As summarizedin Table 1, the effects of substrate concentration, solvent, andincubation time on the yield and enantioselectivity of the hydrolysisreaction can be assessed using a screening approach. In addition, theeffect of altering the alkyl moiety of the acetoacetate (or any β-ketoester) can be assessed using this same approach. The reactions can beconveniently analyzed by chiral HPLC analysis to determine theirconversion ratio and the optical purity of the hydrolyzed product. Atypical chiral HPLC uses, for example, a Chiralcel AD (Daicel ChemicalIndustries) with a mobile phase of hexane: ethanol: trifluoroacetic acid(98:2:0.1%), a flow rate of 1 mL/min and UV detector set to 230 nm.TABLE 1 Substrate Substrate Incubation Conversion (S) Optical (Ester)Concentration Solvent Time ratio Purity (% ee) Ethyl ester  20 mg/ml pH7.0 Buffer   6 hours 40 74 Ethyl ester  10 mg/ml pH 4.0 Buffer 4.5 hours34 85 Ethyl ester  10 mg/ml 90% t-butanol   7 hours 34 92 Ethyl ester100 mg/ml pH 4.0 Buffer  10 hours 10 85 Ethyl ester 100 mg/ml 90%t-butanol  18 hours 38 88 Butyl ester  10 mg/ml 90% t-butanol  10 hours52 70 Isobutyl ester  10 mg/ml 90% t-butanol  16 hours 35 86Trifluoroethyl ester  10 mg/ml 90% t-butanol   1 hour 90 20 Ethyl ester 10 mg/ml 90% acetonitrile  40 hours 42 96 Ethyl ester  10 mg/ml 90%acetone  40 hours 25 97 Ethyl ester  10 mg/ml 90% Isopropanol  40 hours37 94

[0036] Applicants have discovered that using a solvent mixture of anorganic solvent and buffer (or water) in the enzymatic hydrolysisreaction provides better selectivity than buffer alone. The ratio oforganic solvent to buffer ranges from 98:2 or 95:5 to 50:50, preferably,98:2 to 80:20. Applicants have found that in addition to the high yieldand high e.e's obtained using the mixture of organic solvent and buffer,use of the solvent mixture also allows recovery of the enzyme withoutsignificant loss of hydrolase activity. In other words, the enzyme canbe recovered from the reaction mixture, and can be reused for additionalhydrolysis batch runs. In one embodiment, the solvent is selected to beeffective to produce an enantiomeric excess of the desired enantiomer ofthe acid of at least 88%, and preserve at least 90% of the enzymaticactivity of the lipase. Preferably at least 90% of the enzymaticactivity is preserved over four hydrolysis batch cycles. The organicsolvents can be acetonitrile; ketones, for example, acetone; oralcohols, for example, t-butanol and isopropanol. Preferred organicsolvents include acetonitrile, acetone, and t-butanol.

[0037] Buffers useful in the enzymatic reactions of the inventioninclude buffers that have a buffering range of 4 to 8. Useful buffersinclude, for example, acetate buffers, tetraborate buffers, phosphatebuffers, HEPES, Tris-HCl, or citrate buffers. Optimized pH values forthis enzymatic resolution are dependent on the particular enzyme used,and are preferably from 4 to 8, and more preferably from 4 to 6. Apreferred buffer is a 0.2M sodium acetate buffer with a pH of about 4.

[0038] The reaction temperature can be in the range of 5° C. to 70° C.,preferably 15° C. to 37° C.

[0039] The invention provides a method for the convenient recovery ofthe enzyme, the acid 1, and the unreacted ester 5R from the hydrolysisreaction. The enzyme is recovered by for example, filtration and washingwith a mixture of organic solvent and buffer. The enzyme is suitable forreuse in the hydrolysis reaction. The acid 1 and the unreacted ester 5Rcan be recovered from the filtrate. The filtrate is concentrated by, forexample, vacuum distillation to provide a mixture of the acid 1 and theunreacted ester 5R. The mixture is partitioned between an alkalineaqueous solution and an organic solvent such as methyl t-butyl ether(MTBE). After separation of the layers, the unreacted ester 5R isrecovered by concentration of the organic layer. The aqueous solution isacidified and extracted with an organic solvent. This organic extractsare washed with brine and concentrated to furnish the S-acid 1.

[0040] In another embodiment of the invention a method is provided forthe purification of the crude product 1. The ee of the crude product 1can be improved by crystallization from a mixture of solvents. The ee ofthe crude product can be, for example, improved to at least 95%,preferably at least 98%. For example, if the ee of the crude product is88% the ee can preferably be improved to greater than 99% ee with a 65%yield from the crude product. A preferred crystallization solventincludes a mixture of a C, to C₄ alkyl ether and a C₅ to C₇ alkane. Aparticularly preferred solvent includes a mixture of methyl t-butylether (MTBE) and heptane.

[0041] Re-use of the unreacted ester 5R can be achieved by racemizationof 5R to provide additional quantities of the substrate, racemic 5, forthe enzymatic hydrolysis. Any method that provides a racemic mixture of5 can be used for the racemization of 5R including epimerization typereactions and displacement reactions using catalytic amounts ofnucleophiles such as halides. Warming a mixture of the recovered SR andcatalytic amounts of a halide salt is a preferred method of racemizingthe thioacetate ester. In particular, tetraalkyl halides, such astetrabutylammonium bromide, are most preferred as catalysts for theracemization step.

[0042] In an alternative embodiment of the invention, an enzymatichydrolysis of the halo acid ester 4 provides an R α-halo acid 6enantioselectively. This pathway provides a nonracemic chiralintermediate suitable for direct conversion to S α-thioacetate 1.Suitable enzymes and solvents can be screened in the hydrolysis reactionusing a screening approach (See Table 2). For example, Subtilisin BPN(from Sigma), Neutral Protease N (from Amano), and Lipozyme IM (fromNovo Nordisk) give particularly good results using Applicant's reactionconditions. In one embodiment the lipase is immobilized on particles ofa solid support. Suitable solvents for this enzymatic hydrolysisreaction are mixtures of organic solvents and an aqueous buffer. Thepercentage of the organic solvent in the mixture is preferably in therange of 20 to 95%, more preferably in the range of 30% to 60%. Organicsolvents that are useful for this reaction include acetonitrile;ketones, such as acetone and cyclohexanone; cyclic ethers such astetrahydrofuran and 1,4-dioxane; and alcohols such as t-butanol andisopropanol. TABLE 2 Substrate Solvent Conversion (R) Optical Enzyme(ester) % Incubation Ratio Purity (% ee) Lipase-Mucor Ethyl ester pH 7.0buffer  1 hour 38 68 meihei Lipase-Mucor Ethyl ester 95%  5 hour 35 80meihei Cyclohexanone Protease Ethyl ester pH 7.0 buffer  5 hours 45 75subtilisin BPN′ Protease Ethyl ester 30% THF  4 hours 46 84 subtilisinBPN′ Neutral protease Ethyl ester 30% Acetone  2 hours 45 80 Bacillussubtilis Lipase-Mucor Butyl ester 95% 18 hours 60 50 meiheiCyclohexanone Protease Butyl ester 30% THF  4 hours 28 72 subtilisinBPN′ Lipase-Mucor Isobutyl ester 95%  8 hours 25 70 meihei CyclohexanoneProtease Isobutyl ester 30% THF  4 hours 46 80 subtilisin BPN″

[0043] The temperature for this hydrolysis reaction is in the range of5° C. to 70° C., preferably 15° C. to 37° C. The ee of the product 6 ispreferably at least 80%.

[0044] The enantiopure 6 can then be directly converted with inversionof the α-center to 1 using thioacetate salts using the conditionsdescribed above for the racemic α-bromo ester 4.

[0045] The invention provides for recycling of the unreacted S α-haloester 4S from the hydrolysis reaction, similar to the recycling of theα-thioacetyl ester 5R in the first recycling process. Re-use of theunreacted ester 4S can be effected by recovery of and racemization of4S. The unreacted ester 4S can be conveniently recovered by the samemethod that was described above for the unreacted α-thioacetate ester5R. Any method that provides a racemic mixture of additional substrate 4for the enzyme can be used for the racemization step. These methodsinclude epimerization type reactions and displacement reactions usingcatalytic amounts of nucleophiles such as halides. Warming a mixture ofhalo ester 4S and catalytic amounts of a halide salt is a preferredmethod of racemizing the halo ester ester. In particular, tetraalkylhalides, such as tetrabutylammonium bromide, are most preferred ascatalysts for the racemization step.

[0046] Note that while the invention has been described with referenceto Scheme 1, the various compounds of the formulas set forth in theclaims and summary of the invention can be used or made in the processesof the invention. Analogs of the compounds of Scheme 1 required to makethe other compounds of the invention shall be recognizable to those ofordinary skill.

[0047] The following examples further illustrate the present invention,but of course, should not be construed as in any way limiting its scope.

Example 1 Preparation of Ethyl 2-Benzylacetoacetate

[0048] To a 1 L 4-necked round bottom flask that was equipped with anoverhead stirrer, a condenser, an addition funnel and atemperature-probe, was added 207.3 g of ethylacetoacetate. A solution ofsodium ethoxide/ethanol (21 wt. %, 113.1 g) was added at 20-25° C. Thereaction mixture was stirred for 15 minutes. Benzyl chloride (39.14 g,0.3092 mole) was then added through an addition funnel dropwise over 15minutes. The resulting reaction mixture was heated to 80° C. for 2-3hours before cooling to room temperature. Water (300 mL) was added tothe reaction mixture, and the phases were separated in separatoryfunnel. The product was extracted with ethyl acetate (250 mL) threetimes. The combined organic phase was washed with 600 mL of saturatedammonium chloride solution. After drying and evaporation of solvent, theunreacted starting material and product were separated and isolated byfraction distillation under vacuum. The recovered starting materialweighed 137.1 g, 82% recovery yield. Product, ethyl2-benzylacetoacetate, was 64.2 g, 94% yield.

Example 2 Preparation of Ethyl 2-Bromo-3-benzenepropanoate withN-Bromosuccinimide

[0049] To a 2 L 4-necked round bottom flask that was equipped with anoverhead stirrer, an addition funnel, a condenser and atemperature-probe, was added the solution of sodium ethoxide/ethanol(21% wt. %, 161.8 g) at 20-25° C. under nitrogen. Ethyl2-benzylacetoacetate (103.1 g, 0.454 mole) was added dropwise. Theresulting solution was stirred at 20-25° C. for 30 minutes. The reactionmixture was then cooled to −35° C. with a dry ice/acetone bath.N-Bromosuccinimide (NBS; 97.95 g, 0.545 mole) was added portion-wiseover 30 minutes. After the addition was over, the reaction mixture wasstirred at −35° C. for 1 hr before warming up to 20-25° C. and it wasstirred for an additional 2 hrs. The reaction was then quenched withwater (400 mL) and ethyl acetate (600 mL) was added. The layers wereseparated and the product was extracted from aqueous layer with ethylacetate (400 mL×3). The combined organic layers was washed withsaturated sodium bicarbonate twice (400 mL×2), water twice (400 mL×2)and brine (500 mL). After the removal of solvent, the product (crude,132 g) was distilled under reduced pressure. Ethyl2-bromo-3-benzenepropanoate (97.7 g) was obtained at yield 83.7%.

Example 3 Preparation of Ethyl 2-Bromo-3-Benzenepropanoate with(N,N-Dibromo-5,5-dimethylhydantoin

[0050] To a 100 mL 3-necked round bottom flask that was equipped with anoverhead stirrer, an addition funnel, a condenser and atemperature-probe, was added the solution of sodium ethoxide/ethanol(21% wt. %, 0.81 g) at 20-25° C. under nitrogen. Ethyl2-benzylacetoacetate (0.48 g, 2.19 mmol) was added dropwise. Theresulting solution was stirred at 20-25° C. for 30 minutes. The reactionmixture was then cooled to −35° C. with a dry ice/acetone bath.N,N-Dibromo-5,5-dimethylhydantoin (DBDMH; 0.32 g, 1.09 mmol) was addedportion-wise over 30 minutes. After the addition, the reaction mixturewas stirred at −35° C. for 1 hr before warming up to 20-25° C. and itwas stirred for an additional 1 hr. The reaction was then quenched withwater (3 mL) and ethyl acetate (3 mL) was added. The layers wereseparated and the product was extracted from the aqueous layer withethyl acetate (5 mL). The combined organic layers was washed withsaturated sodium bicarbonate (5 mL), and brine (5 mL). After the removalof solvent, ethyl 2-bromo-3-benzenepropanoate (0.46 g) was obtained atyield 81%.

Example 4 Enzymatic Production of Optically Active2-Acetylthio-3-Benzenepropanoic Acid

[0051] Ten (10.0) or 100.0 mg of racemic ethyl2-acetylthio-3-benzenepropanoate was added to 1000 μl of 90%tert-butanol and 10% 200 mM sodium acetate buffer (pH 4.0) whichcontained 10 mg of immobilized lipase from Mucor meihei (Novo). Theglass vessel was tightly capped and stirred for 0-40 hours at roomtemperature. Thirty μl aliquots were removed to ascertain conversion andoptical purity. The aliquots were analyzed using an chiral HPLC column(Chiralcel AD, Daicel Chemical Industries) with a mobile phase ofhexane:ethanol:trifluoroacetic acid 98:2:0.1%. The flow rate is 1ml/minute and detection was at 230 nm. This experiment was alsoperformed in 90% acetonitrile, acetone, and isopropanol as describedabove. Other racemic esters were also evaluated: butyl, isobutyl, andtrifluoroethyl. The results described in Table 1 (above) were obtained.

Example 5 Enzymatic Resolution of Ethyl 2-Acetylthio-3-benzenepropanoate

[0052] To a 500 mL 3-necked round bottom flask that was equipped with anoverhead stirrer and a syringe pump, was added the solution of 0.2MNaOAc (60 mL, pH 5), t-BuOH (240 mL), and then the immobilized lipaseenzyme from Mucor meihei (Novo) (3.0 g). Ethyl2-acetylthio-3-benzenepropanoate (30.0 g, diluted with 15 mL of solvent)was added over 9 hrs using a syringe pump. After the addition, thereaction mixture was stirred at rt for 13 hrs. The enzyme was filteredoff, and washed with t-BuOH and 0.2M NaOAc (2×20 mL, 80:20 v/v). Thesolvent in the filtrate was removed under reduced pressure. Theconcentrated filtrate was mixed with 200 mL MTBE and 150 mL water, andadjusted to pH 7.5 by 2 N NaOH. The layers were separated and the esterwas extracted from the aqueous layer with MTBE (3×100 mL). After theremoval of solvent, ethyl 2-acetylthio-3-benzenepropanoate (19.2 g) wasrecovered, at a 63.9% yield. To the aqueous phase at pH 7.5 was added200 mL MTBE, and the pH was adjusted to pH 2 by 10% H₂SO₄. The layerswere separated and the acid was extracted from aqueous layer with MTBE(3×100 mL). The combined organic layers were washed with brine (2×150mL), and partially distilled at 55° C. under ambient pressure. Afterfurther removal of solvent, (S)-2-acetylthio-3-benzenepropanoic acid(8.3 g) was obtained at a 31.1% yield (ee:88.4%).

Example 6 Crystallization of (S)-2-Acetylthio-3-benzenepropanoic Acid

[0053] The crude (S)-acetylthio-3-benzenepropanoic acid (8.29 g; 88.4%),obtained after solvent removal, was dissolved in MTBE (4 mL, 1.4 mL/g).The solution of acid in MTBE was then heated to 45° C. Heptane (25 mL)was added dropwise to the warm solution until cloudy. The slurry wasseeded with (S)-acetylthio-3-benzenepropanoic acid crystal, and slowlycooled to r.t. without agitation. The addition of heptane was continued(15 mL) with agitation over 30 min. The solid (5.5 g) was collected viavacuum filtration after chilling in an ice/water bath, and washed withcold heptane. The purified (S)-acetylthio-3-benzenepropanoic acid wasassayed by HPLC to determine the purity and % ee (purity 98.2% and ee98.7% at a 66% yield

Example 7 Racemization of Unreacted Ethyl2-Acetylthio-3-benzenepropanoate with Tetrabutylammonium Bromide

[0054] To a 250 mL 3-necked round bottom flask that was equipped with anoverhead stirrer, a condenser and a temperature probe, was added thesolution of ethyl 2-acetylthio-3-benzenepropanoate (47.3 g,R-acid:S-acid=1:1.7) and tetrabutylammonium bromide (6.4 g, 10 mol %).The ester/bromide mixture was heated at 50° C. for 2.5 hrs. The bromidesalt was filtered off, and washed with MTBE (3×100 mL). After solventremoval under reduced pressure, the racemized ethyl2-acetylthio-3-benzenepropanoate (47.6 g, R-acid:S-acid=1:1.2) wasrecovered in essentially 100% yield with some residual solvent.

[0055] Definitions

[0056] The following terms shall have, for the purposes of thisapplication, the respective meanings set forth below.

[0057] Halo. Halo refers to fluoro, chloro, bromo, and iodo. Preferably,the halo moieties are chloro or bromo.

[0058] All publications and references, including but not limited topatents and patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

[0059] While this invention has been described with an emphasis uponpreferred embodiments, it will be obvious to those of ordinary skill inthe art that variations in the preferred devices and methods can be usedand that it is intended that the invention can be practiced otherwisethan as specifically described herein. Accordingly, this inventionincludes all modifications encompassed within the spirit and scope ofthe invention as defined by the claims that follow.

What is claimed:
 1. A method of resolving a racemic mixture of acompound of formula I to obtain a desired enantiomer:

wherein Ar is C₆ or C₁₀ aromatic group that can be substituted with H,C₁ to C₆ alkyl, trifluoromethyl or halo, R₅ is halo or —S—R₁ wherein R₁is H or acetyl, and R₂ is H or C₁ to C₆ alkyl, the method comprising:reacting a compound of formula I wherein the compound is an esterwhereby R₂ is C₁ to C₆ alkyl with a lipase derived from Mucor meihei tostereoselectively hydrolyze the ester bond to produce an acid; andisolating the acid, wherein the reaction is conducted in a solventcomprising 80% to 98% v/v % organic phase and a residue of water phase(which can be buffered).
 2. The method of claim 1, wherein the solventis selected to be effective to (a) produce an enantiomeric excess of thedesired enantiomer of the acid of at least 88% and (b) preserve at least90% of the enzymatic activity of the lipase.
 3. The method of claim 1,wherein the lipase is immobilized on particles of a solid support. 4.The method of claim 1, wherein the organic component of the solventcomprises at least 80% t-butanol, acetonitrile or acetone.
 5. The methodof claim 1, wherein R₁ is acetyl.
 6. A method of stereoselectivelyproducing a desired enantiomer of a compound of formula I:

wherein Ar is C₆ or C₁₀ aromatic group that can be substituted with H,C₁ to C₆ alkyl, trifluoromethyl or halo, R₁ is H or acetyl, and R₂ is Hor C₁ to C₆ alkyl, the method comprising: reacting Ar—CH₂—X, where X isa leaving group, with R₄—C(O)—CH₂—C(O)O—R_(2*), wherein R_(2*) and R₄are independently C₁ to C₆ alkyl; reacting a resulting compound offormula II:

 with a halogenating agent which comprises an N-halo substituted amide,N-halosubstituted imide, N-halosubstituted thioamide, orN-halosubstituted thioimide as the halogenating moiety to produce, withor without an additional hydrolysis of the ester, a compound of formulaIII:

 wherein Y is the leaving group; reacting the compound of formula IIIwith Z—S—R_(1*), wherein R₁ * is acetyl, and Z is K, Na, or other cationto produce a compound of formula I*: and

conducting one of the following stereoselective reactions: (a) (1)reacting the compound of formula III with a hydrolase that isstereoselective for the ester; (2) isolating the desired resulting acid;(3) racemizing residual compound of formula III; and (4) conducting atleast one additional iteration of steps (a)(1) and (a)(2) with theracemized residual compound of formula III, wherein the reacting withZ—S—R₁ * is conducted with stereoselective inversion of the chiralcarbon; or (b) (1) reacting the compound of formula I* with a hydrolasethat is stereoselective for the ester; (2) isolating the desiredresulting acid; (3) racemizing residual compound of formula I*; and (4)conducting at least one additional iteration of steps b(1) and b(2) withthe residual racemized compound of formula I*.
 7. The method of claim 6,further comprising: crystallizing the compound of formula I* to obtainthe compound of formula I* in increased enantiomeric purity.
 8. Themethod of claim 7, wherein the isomeric purity of the compound offormula I* is at least 98% ee.
 9. The method of claim 6, wherein theracemization steps of a(3) and b(3) comprises reacting with a catalyticamount of tetraalkylammonium halide.
 10. The method of claim 6, whereinthe halogenating agent is N,N-dibromo-5,5-dimethylhydantoin.
 11. Themethod of claim 6, wherein the halogenating agent isN,N-dicloro-5,5-dimethylhydantoin.
 12. A method of preparing a compoundof formula II:

wherein R₂ and R₄ are independently C₁ to C₆ alkyl, the methodcomprising: reacting at least five equivalents of R₄—C(O)—CH₂—C(O)O—R₂with ArCH₂Cl wherein Ar is C₆ or C₁₀ aromatic group that can besubstituted with C₁ to C₆ alkyl or halo, wherein the reaction isconducted in a solution consisting essentially of the reactants and nomore than 1.2 molar equivalents of a base source of sodium, potassium,or lithium C₂ to C₆ alkoxide, which can be provided in the correspondingalcohol.
 13. The method of claim 12, wherein the alkoxide concentrationin the base source is at least 3 M.